Project 2 is clinically based. Noonan syndrome (NS) is a relatively common autosomal disorder in which a high percentage of patients have congenital heart defects (CHD), particularly valve abnormalities. PTPN11 mutations cause about 50% of NS. PTPN11 encodes the protein tyrosine phosphatase; SHP-2 and the data are consistent with the hypothesis that most NS mutations result in a gain in SHP-2's function. SPECIFIC AIM 1 will test the hypothesis that PTPN11 mutations are associated with NS with CHD other than pulmonic stenosis as well as with non-syndromic CHD. NS cohorts with other anatomic subtypes and cohorts of subjects with only certain forms of CHD will be assembled and genotyped. Parents of children with only CHD who carry mutations will be genotyped and, if positive, phenotyped for subtle cardiac anomalies. We expect that PTPN11 mutations causing sporadic CHD will be incompletely penetrant. In SPECIFIC AIM 2 we hypothesize that PTPN11 mutations altering phosphotyrosyl-binding domains cause NS by prolonging activation of SHP-2, that specific SHP-2 mutants causing LEOPARD syndrome will have greater gain of function than observed among classic NS mutants, and that mutations causing isolated CHD will result in less gain of function. To test these ideas, mutant SHP-2 proteins will be expressed in eukaryotic cells and tested for phosphatase activity, docking, and MAPK activity. SPECIFIC AIM 3 will test the hypothesis that other genes altering the RAS-MAPK pathway also cause NS. We will study signal transduction in skin fibroblasts derived from NS patients with and without PTPN11 mutations. Next, a candidate gene approach will be used to identify additional NS gene(s). This will be coupled with the use of human multiplex NS kindreds without PTPN11 mutations. SPECIFIC AIM 4's hypothesis is that mice with the D61G mutation will develop pulmonic stenosis and other aspects of NS and that these pathologies will stem from excessive signaling of the EGF pathway. To model accurately the gain-of-function caused by NS-associated PTPN11 mutations, we will construct a D61G "knock-in" into the mouse PTPN11 gene. We will characterize the deranged valvulogenesis during embryonic life and perform epistasis studies. These studies will establish the role of PTPN11 mutations in NS and sporadic CHD, especially those with valve disease, delineate the pathogenesis of PTPN11-related disease, and provide insights into the causes of other genetic forms of NS.